Plaque Formation In Mice Research Articles

Guizhitongluo Tablet inhibits atherosclerosis and foam cell formation through regulating Piezo1/NLRP3 mediated macrophage pyroptosis

BackgroundAtherosclerosis (AS) is the main pathological basis for the development of cardiovascular diseases. Vascular inflammation is an important factor in the formation of AS, and macrophage pyroptosis plays a key role in AS due to its unique inflammatory response. Guizhitongluo Tablet (GZTLT) has shown clinically effective in treating patients with AS, but its mechanism is elusive. PurposeThis study was to determine the effects of GZTLT on atherosclerotic vascular inflammation and pyroptosis and to understand its underlying mechanism. Materials and methodsThe active constituents of GZTLT were analysed by means of UPLC-HRMS. In vivo experiments were performed using ApoE−/− mice fed a high fat diet for 8 weeks, followed by treatment with varying concentrations of GZTLT orally by gavage and GsMTx4 (GS) intraperitoneally and followed for another 8 weeks. Oil red O, Haematoxylin-eosin (HE) and Masson staining were employed to examine the lipid content, plaque size, and collagen fibre content of the mouse aorta. Immunofluorescence staining was utilised to identify macrophage infiltration, as well as the expression of Piezo1 and NLRP3 proteins in aortic plaques. The levels of aortic inflammatory factors were determined using RT-PCR and ELISA. In vitro, foam cell formation in bone marrow-derived macrophages (BMDMs) was observed using Oil Red O staining. Intracellular Ca2+ measurements were performed to detect the calcium influx in BMDMs, and the expression of NLRP3 and its related proteins were detected by Western blot. ResultsThe UPLC-HRMS analysis revealed 31 major components of GZTLT. Our data showed that GZTLT inhibited aortic plaque formation in mice and increased plaque collagen fibre content to stabilise plaques. In addition, GZTLT could restrain the expression of serum lipid levels and suppress macrophage foam cell formation. Further studies found that GZTLT inhibited macrophage infiltration in aortic plaques and suppressed the expression of inflammatory factors. It is noteworthy that GZTLT can restrain Piezo1 expression and reduce Ca2+ influx in BMDMs. Additionally, we found that GZTLT could regulate NLRP3 activation and pyroptosis by inhibiting Piezo1. ConclusionThe present study suggests that GZTLT inhibits vascular inflammation and macrophage pyroptosis through the Piezo1/NLRP3 signaling pathway, thereby delaying AS development. Our finding provides a potential target for AS treatment and drug discovery.

Dietary Evodiamine Inhibits Atherosclerosis-Associated Changes in Vascular Smooth Muscle Cells.

Evodia rutaecarpa (Juss.) Benth is a traditional Chinese medicine. The active ingredient, evodiamine, is a quinolone alkaloid and is found in Evodiae fructus. We investigated the effect of evodiamine on atherosclerosis using LDLR-/- mice fed on a high-fat diet and ox-LDL-induced MOVAS cell lines to construct mouse models and cell-line models. We report a significant reduction in atherosclerotic plaque formation in mice exposed to evodiamine. Our mechanistic studies have revealled that evodiamine can regulate the proliferation, migration, and inflammatory response of and oxidative stress in vascular smooth muscle cells by inhibiting the activation of the PI3K/Akt axis, thus inhibiting the occurrence and development of atherosclerosis. In conclusion, our findings reveal a role for evodiamine in the regulation of vascular smooth muscle cells in atherosclerosis, highlighting a potential future role for the compound as an anti-atherosclerotic agent.

White Button Mushroom (Agaricus bisporus) Supplementation Ameliorates Spatial Memory Deficits and Plaque Formation in an Amyloid Precursor Protein Mouse Model of Alzheimer's Disease.

Alzheimer’s Disease (AD) is characterized by cognitive impairment and the presence of amyloid-β (Aβ) plaques and tau tangles. This study was conducted to assess the effects of white button mushroom (WBM) supplementation on spatial memory and plaque formation in mice with mutations in amyloid (Aβ). Mice with amyloid precursor protein (hAPP) mutations and their wildtype (WT) littermates were fed a 10% white button mushroom (WBM) feed ad libitum three times per week, in addition to their normal diet. Morris water maze (MWM) was conducted at 14 and 32 weeks of age to assess spatial memory and Aβ plaque pathology in the hippocampus was analyzed. Our results showed that hAPP mice on the WBM diet were faster in reaching the platform in the MWM compared to hAPP mice on the control diet at 32 weeks (p < 0.05). Significantly fewer plaque deposits were found in the hippocampi of hAPP mice on the WBM diet compared to those on the control diet at 32 weeks (p < 0.05). Overall, hAPP mice on the WBM diet had improved spatial memory at 32 weeks of age compared to those on the control diet and exhibited fewer amyloid plaques.

0301 Does Orexin Influence Sleep Fragmented Brain Inflammation? A Pilot Study

Abstract Introduction Fragmented sleep occurs when there are repetitive, short interruptions of sleep, resulting in less than six hours of sleep per day. Overall, however, in the United States, sleep fragmentation is reported by 30% of employed adults. Sleep fragmentation may be a risk factor for Alzheimer's disease, and orexin reduction appears effective in reducing amyloid plaque formation in mice with transgenic Alzheimer's disease. Orexin is a neuropeptide that regulates arousal, wakefulness, and appetite. Thus we hypothesized that loss of orexin might also be protective against sleep disruption injury in non-Alzheimer's mice. This study aims to combine sleep fragmentation with orexin loss to see if brain health is severely reduced. Methods The 24 mice in this study were split into four groups: B6 rested, B6 sleep fragmented, orexin knockout rested, orexin knockout sleep fragmented. The sleep fragmented mice were placed on a shaker table for 10½ weeks to initiate chronic sleep loss. The mice were all perfused within five to eight months of birth, and then the brains were cryopreserved and sliced. These sections were immunolabeled with different protein antibodies using immunohistochemistry techniques. The stained brains were either analyzed through microscope stereology counts or computer image analysis. Results Two-way ANOVA analysis for tyrosine hydroxylase, ionized calcium binding adaptor, and vesicular acetylcholine transporter had p&amp;lt;0.05 for the sleep fragmentation variable, showing differences in these antibodies for rested and sleep loss mice. ANOVA for cluster of differentiation 68, cofilin, postsynaptic density protein, and RanBP had p&amp;lt;0.05 for the genotype variable, showing differences in these antibodies for knockout and normal orexin mice. ANOVA for glial fibrillary acidic protein and amyloid-beta had p&amp;lt;0.05 for both variables, showing differences for sleep and orexin levels. There was no ANOVA significance for synapsin. Conclusion Our results show that knocking out orexinergic neurons causes hippocampal tissue damage, dampens the functioning of synapses, and diminishes the ability of the brain to adapt through plasticity and memory. Sleep fragmentation, however, increases phagocytic activity, and harms the acetylcholine and norepinepherine neurotransmitter pathways. When combined, cell communication worsens and the blood brain barrier loses function, resemblant of neurodegenerative diseases. Support (If Any) NIH AG054104; AG064231

Loss of perivascular aquaporin-4 localization impairs glymphatic exchange and promotes amyloid \u03b2 plaque formation in mice

BackgroundSlowed clearance of amyloid β (Aβ) is believed to underlie the development of Aβ plaques that characterize Alzheimer’s disease (AD). Aβ is cleared in part by the glymphatic system, a brain-wide network of perivascular pathways that supports the exchange of cerebrospinal and brain interstitial fluid. Glymphatic clearance, or perivascular CSF-interstitial fluid exchange, is dependent on the astroglial water channel aquaporin-4 (AQP4) as deletion of Aqp4 in mice slows perivascular exchange, impairs Aβ clearance, and promotes Aβ plaque formation.MethodsTo define the role of AQP4 in human AD, we evaluated AQP4 expression and localization in a human post mortem case series. We then used the α-syntrophin (Snta1) knockout mouse model which lacks perivascular AQP4 localization to evaluate the effect that loss of perivascular AQP4 localization has on glymphatic CSF tracer distribution. Lastly, we crossed this line into a mouse model of amyloidosis (Tg2576 mice) to evaluate the effect of AQP4 localization on amyloid β levels.ResultsIn the post mortem case series, we observed that the perivascular localization of AQP4 is reduced in frontal cortical gray matter of subjects with AD compared to cognitively intact subjects. This decline in perivascular AQP4 localization was associated with increasing Aβ and neurofibrillary pathological burden, and with cognitive decline prior to dementia onset. In rodent studies, Snta1 gene deletion slowed CSF tracer influx and interstitial tracer efflux from the mouse brain and increased amyloid β levels.ConclusionsThese findings suggest that the loss of perivascular AQP4 localization may contribute to the development of AD pathology in human populations.

Proprotein Convertase Subtilisin Kexin Type 9 (PCSK9) Beyond Lipids: The Role in Oxidative Stress and Thrombosis.

Proprotein convertase subtilisin/kexin type 9 (PCSK9), mainly secreted in the liver, is a key regulator of cholesterol homeostasis inducing LDL receptors’ degradation. Beyond lipid metabolism, PCSK9 is involved in the development of atherosclerosis, promoting plaque formation in mice and human, impairing the integrity of endothelial monolayer and promoting the events that induce atherosclerosis disease progression. In addition, the PCSK9 ancillary role in the atherothrombosis process is widely debated. Indeed, recent evidence showed a regulatory effect of PCSK9 on redox system and platelet activation. In particular, the role of PCSK9 in the activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox2) system, of MAP-kinase cascades and of CD36 and LOX-1 downstream pathways, suggests that PCSK9 may be a significant cofactor in atherothrombosis development. This evidence suggests that the serum levels of PCSK9 could represent a new biomarker for the occurrence of cardiovascular events. Finally, other evidence showed that PCSK9 inhibitors, a novel pharmacological tool introduced in clinical practice in recent years, counteracted these phenomena. In this review, we summarize the evidence concerning the role of PCSK9 in promoting oxidative-stress-related atherothrombotic process.

The elevation of miR-185-5p alleviates high-fat diet-induced atherosclerosis and lipid accumulation in vivo and in vitro via SREBP2 activation.

Objective: SREBP2, a member of the SREBP family, is a primary regulator of lipid metabolism. In recent years, an increasing number of studies have suggested that miRNAs regulate lipid metabolism-related genes. It was speculated in this study that miRNAs may be implicated in the regulation of lipid accumulation in macrophages by SREBP2 protein.Methods and results: GSE34812, GSE132651 and GSE28829 datasets comprised of atherosclerosis samples were downloaded to explore the gene expression profiles related to the miRNAs and SREBP2, and miR-185-5p was predicted to be a target of SREBP2. The GO annotations and KEGG pathway analysis were adopted for functional classification of differentially expressed genes, and lipid metabolic process was an enriched pathway in atherosclerosis. Besides, the effects of SREBP2 on increasing lipid accumulation were investigated in vivo using miR-185-5p mimic/apoE−/− mice and miR-185-5p NC/apoE−/− mice. All mice fed with a HFD suffered from atherosclerosis. Moreover, the effects of miR-185-5p on atherosclerotic plaque formation in mice were analyzed. An in vitro assay was also performed to determine the effect of miR-185-5p on ox-LDL-stimulated RAW 264.7 macrophages. Finally, miR-185-5p mimic was transfected into cultured macrophages. The results showed that the miR-185-5p elevation might regulate lipid accumulation in mice by targeting SREBP2. Furthermore, miR-185-5p mimic repressed the activation of SREBP1, SREBP2, LDLR, SCD-1, HMGCR as well as NLRP3, IL-1β, TNF-α in HFD fed mice or ox-LDL-stimulated macrophages.Conclusions: our study demonstrated that miR-185-5p effectively alleviates atherosclerosis and lipid accumulation by regulating the miR-185-5p/SREBP2 axis.

Abstract 12444: Smooth Muscle-Specific Deletion of Perk Prevents Atherosclerotic Plaque Formation in Mice

Introduction: Lineage tracing and single cell RNA sequencing (scRNA-Seq) studies have identified a subset of phenotypically modulated vascular smooth muscle cells (SMCs) that appear with atherosclerotic plaque formation in hyperlipidemic mice. These modulated SMCs (mSMCs) are de-differentiated, and increase expression of macrophage, fibroblast and stem cell markers. We previously showed that SMCs exposed to free cholesterol in vitro activate an endoplasmic reticulum unfolded protein response (UPR) and assume an mSMC phenotype, and blocking Perk specifically abolishes this SMC modulation. Methods: Here, we sought to define the role of Perk in atherosclerotic plaque formation using SMC-specific Perk knockout in C57BL6 mice ( Perk SMC-KO ) and inducing hyperlipidemia by PSCK9 DY overexpression and a high fat diet for 12 weeks. Results: Aortic Oil Red O staining and en face aortic analysis revealed no plaque formation in the Perk SMC-KO aorta, with the exception of some small plaques in the root, whereas the wild-type (WT) aortas had extensive plaque, especially in the aortic arch (N = 10, p&lt;0.0001, Fig. 1A ). scRNA-Seq analysis of aortic tissue from the root to the arch from these mice showed no major differences in cell clusters between Perk SMC-KO and WT mice at baseline ( Fig. 1B ). In hyperlipidemic WT mice, SMC-derived cluster 8 expands and a new cluster 7 appears (circled in red, Fig. 1B ). In Perk SMC-KO mice, cluster 8 is similarly expanded with hyperlipidemia, while cluster 7 is completely absent ( Fig. 1B , bottom right). Cluster 7 in the WT atherosclerotic aortas is the only cluster with Perk expression and has significantly increased expression of macrophage markers and a subset of fibroblast-specific markers when compared to cluster 8. Conclusions: These data indicate that SMC phenotypic modulation plays a major role in atherosclerotic plaque formation and Perk is a key regulator of the SMC modulation that specifically contributes to atherosclerotic plaque burden.

ALDH4A1 is an atherosclerosis auto-antigen targeted by protective antibodies.

Cardiovascular disease (CVD) is the leading cause of mortality in the world, with most CVD-related deaths resulting from myocardial infarction or stroke. The main underlying cause of thrombosis and cardiovascular events is atherosclerosis, an inflammatory disease that can remain asymptomatic for long periods. There is an urgent need for therapeutic and diagnostic options in this area. Atherosclerotic plaques contain autoantibodies1,2, and there is a connection between atherosclerosis and autoimmunity3. However, the immunogenic trigger and the effects of the autoantibody response during atherosclerosis are not well understood3-5. Here we performed high-throughput single-cell analysis of the atherosclerosis-associated antibody repertoire. Antibody gene sequencing of more than 1,700 B cells from atherogenic Ldlr-/- and control mice identified 56 antibodies expressed by in-vivo-expanded clones of B lymphocytes in the context of atherosclerosis. One-third of the expanded antibodies were reactive against atherosclerotic plaques, indicating that various antigens in the lesion can trigger antibody responses. Deep proteomics analysis identified ALDH4A1, a mitochondrial dehydrogenase involved in proline metabolism, as a target antigen of one of these autoantibodies, A12. ALDH4A1 distribution is altered during atherosclerosis, and circulating ALDH4A1 is increased in mice and humans with atherosclerosis, supporting the potential use of ALDH4A1 as a disease biomarker. Infusion of A12 antibodies into Ldlr-/- mice delayed plaque formation and reduced circulating free cholesterol and LDL, suggesting that anti-ALDH4A1 antibodies can protect against atherosclerosis progression and might have therapeutic potential in CVD.

Endogenous oxidized phospholipids reprogram cellular metabolism and boost hyperinflammation

Abstract Pathogen-associated molecular patterns (PAMPs) have the capacity to couple inflammatory gene expression to changes in macrophage metabolism, both of which influence subsequent inflammatory activities. Similar to their microbial counterparts, several self-encoded damage-associated molecular patterns (DAMPs) induce inflammatory gene expression. However, whether this symmetry in host responses between PAMPs and DAMPs extends to metabolic shifts is unclear. Here we report that the self-encoded oxidized phospholipid oxPAPC alters the metabolism of macrophages exposed to lipopolysaccharide (LPS). While cells activated by LPS rely exclusively on glycolysis, macrophages exposed to oxPAPC also use mitochondrial respiration, feed the Krebs cycle with glutamine and favor the accumulation of oxaloacetate in the cytoplasm: this metabolite potentiates IL-1beta production, resulting in hyperinflammation. Similar metabolic adaptions occur in vivo in hypercholesterolemic mice and human subjects. Drugs that interfere with oxPAPC-driven metabolic changes reduce atherosclerotic plaque formation in mice, thereby underscoring the importance of DAMP-mediated activities in pathophysiological conditions.

Endogenous oxidized phospholipids reprogram cellular metabolism and boost hyperinflammation.

Pathogen-associated molecular patterns (PAMPs) have the capacity to couple inflammatory gene expression to changes in macrophage metabolism, both of which influence subsequent inflammatory activities. Similar to their microbial counterparts, several self-encoded damage-associated molecular patterns (DAMPs) induce inflammatory gene expression. However, whether this symmetry in host responses between PAMPs and DAMPs extends to metabolic shifts is unclear. Here we report that the self-encoded oxidized phospholipid oxPAPC alters the metabolism of macrophages exposed to lipopolysaccharide (LPS). While cells activated by LPS rely exclusively on glycolysis, macrophages exposed to oxPAPC also use mitochondrial respiration, feed the Krebs cycle with glutamine and favor the accumulation of oxaloacetate in the cytoplasm: this metabolite potentiates IL-1β production, resulting in hyperinflammation. Similar metabolic adaptions occur in vivo in hypercholesterolemic mice and human subjects. Drugs that interfere with oxPAPC-driven metabolic changes reduce atherosclerotic plaque formation in mice, thereby underscoring the importance of DAMP-mediated activities in pathophysiological conditions.

MiR-133b Downregulation Reduces Vulnerable Plaque Formation in Mice with AS through Inhibiting Macrophage Immune Responses

Atherosclerosis (AS) is a chronic inflammatory disease characterized by accumulating deposition of lipids in the arterial intima. Notably, macrophages participate centrally in the pathogenesis of this deadly disease. In this study, we established AS mouse models in order to investigate the effect of microRNA-133b (miR-133b) on vulnerable plaque formation and vascular remodeling in AS and explore the potential functional mechanisms. The expression of miR-133b was altered or the Notch-signaling pathway was blocked in the AS mouse models in order to evaluate the proliferation, migration, and apoptosis of macrophages. It was observed that miR-133b was upregulated in AS, which might target MAML1 to regulate the Notch-signaling pathway. AS mice with downregulated miR-133b or inhibited Notch-signaling pathway presented with a reduced AS plaque area, a decreased positive rate of macrophages, and an increased positive rate of vascular smooth muscle cells. Moreover, Notch-signaling pathway blockade or miR-133b downregulation inhibited the macrophage viability and migration and accelerated the apoptosis. This study provides evidence that downregulated miR-133b expression may inhibit the immune responses of macrophages and attenuate the vulnerable plaque formation and vascular remodeling in AS mice through the MAML1-mediated Notch-signaling pathway, highlighting miR-133b as a novel therapeutic target for AS.

Reduced Plasma Kallistatin Is Associated With the Severity of Coronary Artery Disease, and Kallistatin Treatment Attenuates Atherosclerotic Plaque Formation in Mice.

BackgroundKallistatin exerts beneficial effects on organ injury by inhibiting oxidative stress and inflammation. However, the role of kallistatin in atherosclerosis is largely unknown. Here, we investigated the role and mechanisms of kallistatin in patients with coronary artery disease (CAD), atherosclerotic plaques of apoE−/− mice, and endothelial activation.Methods and ResultsPlasma kallistatin levels were analyzed in 453 patients at different stages of CAD. Kallistatin levels were significantly lower in patients with CAD and negatively associated with CAD severity and oxidative stress. Human kallistatin cDNA in an adenoviral vector was injected intravenously into apoE−/− mice after partial carotid ligation, with or without nitric oxide synthase inhibitor (Nω‐nitro‐L‐arginine methyl ester) or sirtuin 1 inhibitor (nicotinamide). Kallistatin gene delivery significantly reduced macrophage deposition, oxidative stress, and plaque volume in the carotid artery, compared with control adenoviral injection. Kallistatin administration increased endothelial nitrous oxide synthase, sirtuin 1, interleukin‐10, superoxide dismutase 2, and catalase expression in carotid plaques. The beneficial effects of kallistatin in mice were mitigated by Nω‐nitro‐L‐arginine methyl ester or nicotinamide. Furthermore, human kallistatin protein suppressed tumor necrosis factor‐α–induced NADPH oxidase activity and increased endothelial nitrous oxide synthase and sirtuin 1 expression in cultured human endothelial cells. These effects were also abolished by Nω‐nitro‐L‐arginine methyl ester or nicotinamide.ConclusionsThis was the first study to demonstrate that reduced plasma kallistatin levels in patients are associated with CAD severity and oxidative stress. Kallistatin treatment prevents carotid atherosclerotic plaque formation in mice by stimulating the sirtuin 1/endothelial nitrous oxide synthase pathway. These findings indicate the potential protective effects of kallistatin on atherosclerosis in human subjects and mouse models.

Pro-atherogenic high fat diet accelerates tumor growth in mice through activation of innate immunity

Inflammation is a physiologic protective process triggered in response to injury or infection. When unresolved, inflammation often promotes the development of chronic pathologies such as cancer or atherosclerosis. In atherosclerosis, hypercholesterolemia-associated monocytosis drives plaque formation in mice. Epidemiologic and experimental studies have shown the involvement of dyslipidiemia and low-grade inflammation on the development of multiple cancers. Surprisingly, the link between atherosclerosis and cancer has not been studied in details. The objective of our study was to question whether a pro-atherogenic high fat diet (HFD) would modify the development of solid tumors. Our experimental approach was to feed mice susceptible to atherosclerosis with a classical HFD (15% fat, 1.25% cholesterol) or a chow diet for 2 weeks before induction of tumor development. We monitored tumor development every two days and analyzed the mechanistic events by in vitro and in vivo experiments. We found that the proatherogenic HFD accelerated the growth of several solid tumors (B16F10 and TC1) in C57Bl6 mice, independently of cholesterol levels. In contrast, the HFD induced a significant increase of myeloid cells number in circulation and in the tumor microenvironment (flow cytometry). We isolated (Miltenyi) and analyzed the phenotype (qPCR and ELISA) of tumor-infiltrating leukocytes (TILs). We found a rapid upregulation of IL-1beta and VEGFa expression and a downregulation of several inflammatory cytokines. The supernatant of TILs isolated from mice under HFD and cultured for 48 hours, failed to reduce tumor cell proliferation in vitro (tritiated thymidine) and absence of IL-1beta, or selective depletion of neutrophils protected mice against tumor acceleration under HFD. These data show that in the context of a proaterogenic HFD, neutrophils accumulate in the tumor microenvironment and switch toward an immunosuppressive phenotype, thus promoting the fast growing of tumors.

Diesel engine exhaust accelerates plaque formation in a mouse model of Alzheimer\u2019s disease

BackgroundIncreasing evidence from toxicological and epidemiological studies indicates that the central nervous system is an important target for ambient air pollutants. We have investigated whether long-term inhalation exposure to diesel engine exhaust (DEE), a dominant contributor to particulate air pollution in urban environments, can aggravate Alzheimer’s Disease (AD)-like effects in female 5X Familial AD (5XFAD) mice and their wild-type female littermates. Following 3 and 13 weeks exposures to diluted DEE (0.95 mg/m3, 6 h/day, 5 days/week) or clean air (controls) behaviour tests were performed and amyloid-β (Aβ) plaque formation, pulmonary histopathology and systemic inflammation were evaluated.ResultsIn a string suspension task, assessing for grip strength and motor coordination, 13 weeks exposed 5XFAD mice performed significantly less than the 5XFAD controls. Spatial working memory deficits, assessed by Y-maze and X-maze tasks, were not observed in association with the DEE exposures. Brains of the 3 weeks DEE-exposed 5XFAD mice showed significantly higher cortical Aβ plaque load and higher whole brain homogenate Aβ42 levels than the clean air-exposed 5XFAD littermate controls. After the 13 weeks exposures, with increasing age and progression of the AD-phenotype of the 5XFAD mice, DEE-related differences in amyloid pathology were no longer present. Immunohistochemical evaluation of lungs of the mice revealed no obvious genetic background-related differences in tissue structure, and the DEE exposure did not cause histopathological changes in the mice of both backgrounds. Luminex analysis of plasma cytokines demonstrated absence of sustained systemic inflammation upon DEE exposure.ConclusionsInhalation exposure to DEE causes accelerated plaque formation and motor function impairment in 5XFAD transgenic mice. Our study provides further support that the brain is a relevant target for the effects of inhaled DEE and suggests that long-term exposure to this ubiquitous air pollution mixture may promote the development of Alzheimer’s disease.

P692Palmitoylethanolamide promotes an anti-inflammatory macrophage phenotype and attenuates atherosclerotic plaque formation in mice

P692Palmitoylethanolamide promotes an anti-inflammatory macrophage phenotype and attenuates atherosclerotic plaque formation in mice

Abstract 622: Development of an Antioxidized Phospholipid Neutralizing Antibody as an Atheroprotective Therapeutic Agent

Atherosclerosis is a chronic disease of the arterial wall, which contributes to &gt;40% of all deaths in developed societies. Widespread use of statins and life style modifications within developed nations have resulted in modest reductions in the incidences and severity of atherosclerotic disease, but as yet there is no clear evidence that statins can prevent late stage clinical consequences of atherosclerosis including plaque rupture with possible myocardial infarction or stroke. Atherosclerosis is a complex disease characterized by two key processes, lipid deposition and inflammation within the arterial wall, and oxidative stress providing the link between the two. Oxidized phospholipids (OxPL), such as oxidized PAPC (1-palmitoil-2-arachodonoyl-sn-glycero-3-phosphorylcholine) and its derivatives are the biologically active components of minimally oxidized LDL, whose role in cardiovascular diseases is well recognized. Atherosclerotic ApoE knockout (KO) mice fed a high fat Western diet develop antibodies (Abs) to OxPL, and hybridoma B cell lines producing natural auto-Abs against OxPL have been successfully generated and characterized. However, as yet, no studies have been reported demonstrating that treatment with OxPL neutralizing Abs can be used to prevent or reverse atherosclerosis. Of major importance, we recently generated an anti-OxPL auto-Ab (10C12) that demonstrates potent OxPL neutralizing activity in vitro. Sequence analysis showed that 10C12 Ab is structurally different from previously described anti-OxPL EO6 type neutralizing Abs. Moreover, we found that 10C12 Ab injections (IP, twice a week) in ApoE KO mice fed a Western diet for four weeks resulted in: 1) high circulating titers of Abs to OxPL as measured by ELISA; 2) significantly decreased accumulation of macrophages within the brachiocephalic artery, a site of advanced plaque formation in mice; and 3) a decreased OxPL-induced inflammatory response following IP injection of OxPL as determined by analysis of inflammatory gene expression in abdominal lavage cells. Results of our work give insight into the role of OxPL in atherosclerosis and lead to potential novel therapeutic approaches to treat it or prevent clinical complications of atherosclerosis.

Elevated arterial wall permeability may cause vulnerable plaque formation in mice

Elevated arterial wall permeability may cause vulnerable plaque formation in mice

Role of Extracellular RNA in Atherosclerotic Plaque Formation in Mice

Atherosclerosis and vascular remodeling after injury are driven by inflammation and mononuclear cell infiltration. Extracellular RNA (eRNA) has recently been implicated to become enriched at sites of tissue damage and to act as a proinflammatory mediator. Here, we addressed the role of eRNA in high-fat diet-induced atherosclerosis and neointima formation after injury in atherosclerosis-prone mice. The presence of eRNA was revealed in atherosclerotic lesions from high-fat diet-fed low-density lipoprotein receptor-deficient (Ldlr(-/-)) mice in a time-progressive fashion. RNase activity in plasma increased within the first 2 weeks (44±9 versus 70±7 mU/mg protein; P=0.0012), followed by a decrease to levels below baseline after 4 weeks of high-fat diet (44±9 versus 12±2 mU/mg protein; P<0.0001). Exposure of bone marrow-derived macrophages to eRNA resulted in a concentration-dependent upregulation of the proinflammatory mediators tumor necrosis factor-α, arginase-2, interleukin-1β, interleukin-6, and interferon-γ. In a model of accelerated atherosclerosis after arterial injury in apolipoprotein E-deficient (ApoE(-/-)) mice, treatment with RNase1 diminished the increased plasma level of eRNA evidenced after injury. Likewise, RNase1 administration reduced neointima formation in comparison with vehicle-treated ApoE(-/-) controls (25.0±6.2 versus 46.9±6.9×10(3) μm(2), P=0.0339) and was associated with a significant decrease in plaque macrophage content. Functionally, RNase1 treatment impaired monocyte arrest on activated smooth muscle cells under flow conditions in vitro and inhibited leukocyte recruitment to injured carotid arteries in vivo. Because eRNA is associated with atherosclerotic lesions and contributes to inflammation-dependent plaque progression in atherosclerosis-prone mice, its targeting with RNase1 may serve as a new treatment option against atherosclerosis.

Langerin neg conventional dendritic cells produce IL-23 to drive psoriatic plaque formation in mice

Psoriasis is an autoinflammatory skin disease of unknown etiology. Topical application of Aldara cream containing the Toll-like receptor (TLR)7 agonist Imiquimod (IMQ) onto patients induces flares of psoriasis. Likewise, in mice IMQ triggers pathological changes closely resembling psoriatic plaque formation. Key cytokines like IL-23 and type-I IFN (IFN-I), both being produced mainly by dendritic cells (DCs), have been implicated in psoriasis. Although plasmacytoid DCs (pDCs) are the main source of IFNα and thought to initiate disease, conventional DCs (cDCs) appear to maintain the psoriatic lesions. Any role of cDCs during lesion formation remains elusive. Here, we report that selective activation of TLR7 signaling specifically in CD11c(+) DCs was sufficient to induce psoriasiform skin disease in mice. Intriguingly, both pDCs and the IFN-I pathway were dispensable for the development of local skin inflammation. Selective TLR7 triggering of Langerin(+) DCs resulted in attenuated disease, whereas their depletion did not alter the severity of skin lesions. Moreover, after IMQ-painting, IL-23 was exclusively produced by Langerin(neg) DCs in vivo. In conclusion, TLR7-activated Langerin(neg) cDCs trigger psoriatic plaque formation via IL-23-mediated activation of innate IL-17/IL-22-producing lymphocytes, independently of pDCs or IFN-I. These results suggest therapeutic targeting of IL-23 production by cDCs to refine current treatment strategies for psoriasis.